This application requests a Roche Genome Sequencer FLX (GS FLX), developed by 454 Sequencing for placement in the Johns Hopkins Bloomberg School of Public Health. This instrument generates over one million unique high quality sequencing reads with an average of 350 to 400 bases pairs in length within a ten hour instrument run time. The total average yield per instrument run is 400 to 500 megabases. The instrument will be used by a diverse group of investigators addressing questions related to the major disease epidemics confronting the world today. Representative studies include those of primary resistance to antiretroviral therapy for HIV among drug users and among infants born to mothers receiving prophylactic antiretroviral therapy. These studies will evaluate the usefulness of this technology for guiding initial antiretroviral therapy in those distinct settings. The genetic basis for resistance to anti-tuberculosis agents will be studied among tubercle bacilli lacking the traditional resistance mechanisms. Differences among malaria parasites that can or cannot complete the transmission cycle in mosquitoes will be defined using this instrument, as will the mosquito?s innate immune response to the presence of malaria. The biology of cancer may be elucidated by studies identifying compensatory mutations in yeast knockout strains, with many secondary mutations yielding phenotypes observed in cancer cell lines. Similarly studies will characterize distinct genetic features of those hepatitis B virus variants identified in individuals with hepatocellular carcinoma. All of these studies have in common the need to detect either rare mutations in a large and diverse gene pool or mutations in a large genome that account for a distinct phenotype. In all cases that need is addressed by the high throughput sequencing capability of the Genome Sequencer FLX system. Analytical tools provided with the instrument will aid in the proposed analyses, but this project also takes advantage of a bioinformatics core established specifically for purposes of genome analysis at Johns Hopkins University. Finally the instrument will be housed in a pre-existing core facility originally established for array analysis but with the instrumentation and available expertise to exploit the capabilities of high throughput sequencing.